During the embryonic period of CNS development NSCs are considered to be the proliferating cellular compartment in the neuroepithelium that both expands itself through self-renewal via symmetrical divisions and generates lineage-restricted progenitors via asymmetrical divisions. Progenitor progeny also divide both symmetrically and asymmetrically and ultimately differentiate into the neural phenotypes composing different stages of CNS development. Although the seminal biology of NSCs has come under intensive investigation, there is still no consensus regarding exactly which cells are actually NSCs, since specific markers do not exist. So, neuroepithelial cells are widely used as the primary source of NSCs, and often as if they all were NSCs. Thus, there is a general consensus that further elucidation of NSC biology first requires their identification since this could provide direct experimental access to them for prospective rather than retrospective investigation. Previously, we surface-phenotyped neuroepithelial cells at the onset of neurogenesis, aiming to identify cells not yet overtly committed to neural lineages as a logical source of putative NSCs. Such lineage-negative neuroepithelial cells predominated, accounting for 90% of the dissociated population. When cultured at clonal density in defined medium with basic fibroblast growth factor (bFGF) these cells exhibited four stereotypical clonal expansion stages: 1) proliferation without differentiation (self-renewal), expanding the compartment of undifferentiated neuroepithelial cells, 2) proliferation with frequent apoptosis, maintaining undifferentiated neuroepithelial cells, 3) neurogenic, producing the earliest-born cortical neurons and 4) multi-potential, sequentially generating multiple neuronal and glial progenitors in their correct chronological order, paralleling their developmental appearance in vivo. Further research revealed a clear lineage progression in these seminal properties, which was related to developmental up-regulation of both an amino acid transporter and a third FGF receptor (FGFR2). These results demonstrate that lineage-negative neuroepithelial cells do indeed exhibit seminal properties typically attributed to the NSC phenotype, all of which is well-preserved in vitro. Preservation of these seminal properties implies a dominant role for cell-intrinsic mechanisms that can be accessed under experimentally controlled conditions.[unreadable] [unreadable] During FY2007, we focused to varying degrees on the four specific aims outlined above. We have incorporated a number of other markers used by others in the field of neural stem cell biology in an effort to reveal how our multi-epitope phenotyping strategy compares with, and is related to, those used elsewhere. Since multi-lineage-negative neuroepithelial cells with the seminal properties of NSCs largely disappear at the onset of neurogenesis, it is evident that one or more of their progenitor progeny maintain the NSC phenotype. We found that NSC-derived progenitors co-expressing JONES- and A2B5- reactive complex gangliosides (neuroglial progenitors, NGPs) also express the trisaccharide Lewis X (LeX) antigen. LeX has become increasingly used as a marker of "neural stem/progenitor" cells. Thus, co-expression of LeX by JONES+ A2B5+ NGPs provides an important and insightful link between our phenotyping strategy and that used by others. Furthermore, LeX+ JONES+ A2B5+ NGPs predominate throughout corticogenesis. Preliminary results show that these progenitors can self-renew and differentiate, first generating neuronal progenitors, which develop into transient and permanent neuron populations composing the cortex. Later, they generate astroglial (LeX+ JONES+ A2B5+ GFAP+) and oligoglial progenitor progeny (LeX+ JONES+ A2B5+ O4+). Therefore, LeX+ JONES+ A2B5+ NGPs derived from lineage-negative NSCs maintain the NSC phenotype, exhibiting similar, but not identical properties as lineage-negative NSCs. Lineage-negative NSCs predominant at the onset of neurogenesis produce transient neural phenotypes, while LeX+ JONES+ A2B5+ NGPs abundant thereafter generate permanent neuronal and glial phenotypes. Other markers tested thus far, including CD133 and Sox2, are expressed by NSCs and LeX+ JONES+ A2B5+ NGPs to varying degrees and so may considered persistent, though not all-inclusive markers of the NSC phenotype. We plan to extend these studies by sorting the two NSC phenotypes during neurogenesis to assess their relative contributions to the different cortical layers. The predominance of LeX+ JONES+ A2B5+ NGPs during neurogenesis beginning at embryonic day 14 and thereafter strongly suggests that these cells make the larger contribution to seeding the cortex with permanent neural phenotypes.[unreadable] [unreadable] We have further characterized the earliest-born neurons derived from lineage-negative NSCs and LeX+ JONES+ A2B5+ NGPs. Those generated first are calbindin+ calretinin+ reelin- choline acetyltransferase+ (ChAT+), identifying them as cholinergic pioneer neurons. ChAT+ projections of pioneer neurons, which are transiently expressed, themselves transiently project into the proliferating neuroepithelium. Hence, secretion of the neurotransmitter acetylcholine might regulate the seminal properties of lineage-negative NSCs and LeX+ JONES+ A2B5+ NGPs residing there. Both NSCs and NGPs express multiple cholinergic muscarinic receptors whose activation triggers neurogenesis in vitro. Blockade of muscarinic receptors in vivo attenuates generation of both pioneer neurons and NGPs from NSCs. Collectively, these results indicate that transiently-expressed cholinergic pioneer neurons may regulate the seminal properties of neuroepithelial cells with NSC biologies. The next type of neuron differentiating at the onset of neurogenesis is the calbindin+ calretinin+ reelin+ Cajal-Retzius neuron, which is derived from asymmetrical division of NGPs. These neurons are also cholinergic and, from results published elsewhere, help to regulate proper cortical layering via their secretion of reelin. The discovery that they are also cholinergic indicates that their role in regulating corticogenesis may also include cholinergic signaling. This remains to be determined.[unreadable] [unreadable] We have collaborated with the Developmental Genetics Section, NINDS to carry out cDNA microarray and microRNA gene expression profiling during the earliest stages of neurogenesis using FACS-sorted NSCs, NGPs and neuronal progenitors (NPs). Microarray analyses revealed erythroid precursors and neural crest stem cells as minor contaminants of the NSC population. Thus far, we have eliminated erythroid precursors and plan to eliminate neural crest stem cells in order to obtain a pristine gene expression profile of the earliest neuroepithelial cells with the seminal properties of NSCs. We will then compare this to profiles obtained from NGPs, which, as detailed above, also exhibit similar, though not identical seminal properties. These profiles should resolve gene expression patterns of two primary NSC phenotypes. Profiling of NPs has revealed a number of genes whose deletions are associated with cortical defects, according to the published literature. These results suggest that the early-born pioneer neurons, though transient, do play critical roles in regulating cortical development.[unreadable] [unreadable] Injection of FACS-sorted, particle-loaded NSCs and NGPs into different forebrain regions of rodent hosts in collaboration with the Laboratory of Functional and Molecular Imaging has shown that both populations survive, migrate and differentiate into multiple neural phenotypes. Future experiments will target younger hosts and use more enduring labeling protocols in order to avoid misinterpretation of bystander cells becoming labeled following the death of labeled donor cells.